Therapeutic anti-cytomegalovirus compounds

ABSTRACT

The present invention provides synthetic compounds, antibodies that recognize and bind to these compounds, polynucleotides that encode these compounds, and immune effector cells raised in response to presentation of these epitopes. The invention further provides methods for inducing an immune response and administering immunotherapy to a subject by delivering the compositions of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 09/812,079 filed Mar. 19, 2001, now U.S. Pat. No. 6,579,970, whichclaims priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication Ser. Nos. 60/191,050 and 60/254,989, filed Mar. 21, 2000 andDec. 12, 2000 respectively. The entire contents of these applicationsare hereby incorporated by reference into the present disclosure.

TECHNICAL FIELD

The invention relates to the field of therapeutic compounds usefulagainst Cytomegalovirus (“CMV”) infections.

BACKGROUND OF THE INVENTION

The recognition of antigenic epitopes presented by molecules of theMajor Histocompatibility Complex (MHC) plays a central role in theestablishment, maintenance and execution of mammalian immune responses.T cell surveillance and recognition of peptide antigens presented bycell surface MHC molecules expressed by somatic cells and antigenpresenting leukocytes functions to control invasion by infectiousorganisms such as viruses, bacteria, and parasites. In addition it hasnow been demonstrated that antigen-specific cytotoxic T lymphocytes(CTLs) can recognize certain cancer cell antigens and attack cellsexpressing these antigens. This T cell activity provides a basis fordeveloping novel strategies for anti-cancer vaccines. Furthermore,inappropriate T cell activation plays a central role in certaindebilitating autoimmune diseases such as rheumatoid arthritis, multiplesclerosis, and asthma. Thus presentation and recognition of antigenicepitopes presented by MHC molecules play a central role in mediatingimmune responses in multiple pathological conditions.

Tumor specific T cells, derived from cancer patients, will bind and lysetumor cells. This specificity is based on their ability to recognizeshort amino acid sequences (epitopes) presented on the surface of thetumor cells by MHC class I and, in some cell types, class II molecules.These epitopes are derived from the proteolytic degradation ofintracellular proteins called tumor antigens encoded by genes that areeither uniquely or aberrantly expressed in tumor or cancer cells.

The availability of specific anti-tumor T cells has enabled theidentification of tumor antigens and subsequently the generation ofcancer vaccines designed to provoke an anti-tumor immune response.Anti-tumor T cells are localized within cancer patients, including inthe blood (where they can be found in the peripheral blood mononuclearcell fraction), in primary and secondary lymphoid tissue, e.g., thespleen, in ascites fluid in ovarian cancer patients (tumor associatedlymphocytes or TALs) or within the tumor itself (tumor infiltratinglymphocytes or TILs). Of these, TILs have been the most useful in theidentification of tumor antigens and tumor antigen-derived peptidesrecognized by T cells.

Conventional methods to generate TILs involve mincing tumor biopsytissue and culturing the cell suspension in vitro in the presence of theT cell growth factor interleukin 2 (IL-2). Over a period of severaldays, the combination of the tumor cells and IL-2 can stimulate theproliferation of tumor specific T cells at the expense of tumor cells.In this way, the T cell population is expanded. The T cells derived fromthe first expansion are subsequently mixed with either mitomycinC-treated or irradiated tumor cells and cultured in vitro with IL-2 topromote further proliferation and enrichment of tumor reactive T cells.After several rounds of in vitro expansion, a potent anti-tumor T cellpopulation can be recovered and used to identify tumor antigens viaconventional but tedious expression cloning methodology. Kawakami Y. etal. (1994) Proc. Natl. Acad. Sci. USA 91(9):3515–3519.

This currently employed methodology used to generate tumor specific Tcells in vitro is unreliable and the antigens identified by this methoddo not necessarily induce an anti-tumor immune response. Numerousexperiments demonstrate that the encounter of antigens by mature T cellsoften results in the induction of tolerance because of ignorance, anergyor physical deletion. Pardoll (1998) Nature Med. 4(5):525–531.

The ability of a particular peptide to function as a T cell epitoperequires that it bind effectively to the antigen presenting domain of anMHC molecule and also that it display an appropriate set of amino acidsthat can be specifically recognized by a T cell receptor molecule. Whileit is possible to identify natural T cell epitopes derived fromantigenic polypeptides, these peptide epitopes do not necessarilyrepresent antigens that are optimized for inducing a particular immuneresponse. In fact, it has been shown that it is possible to improve theeffectiveness of natural epitopes by introducing single amino ormultiple acids substitutions that alter their sequence (Valmori et al.(2000) J. Immunol 164(2):1125–1131). Thus, delivery of carefullyoptimized synthetic peptide epitopes has the potential to provide animproved method to induce a useful immune response.

The introduction into an animal of an antigen has been widely used forthe purposes of modulating the immune response, or lack thereof, to theantigen for a variety of purposes. These include vaccination againstpathogens, induction of an immune response to a cancerous cell,reduction of an allergic response, reduction of an immune response to aself antigen that occurs as a result of an autoimmune disorder,reduction of allograft rejection, and induction of an immune response toa self antigen for the purpose of contraception.

In the treatment of cancer, a variety of immunotherapeutic approacheshave been taken to generate populations of cytotoxic T lymphocytes whichspecifically recognize and lyse tumor cells. Many of these approachesdepend in part on identifying and characterizing tumor-specificantigens.

More recently, certain pathogen- and tumor-related proteins have beenimmunologically mimicked with synthetic peptides whose amino acidsequence corresponds to that of an antigenic determinant domain of thepathogen- or tumor-related protein. Despite these advances, peptideimmunogens based on native sequences generally perform less thanoptimally with respect to inducing an immune response. Thus, a needexists for modified synthetic antigenic peptide epitopes with enhancedimmunomodulatory properties. This invention satisfies this need andprovides related advantages as well.

DISCLOSURE OF THE INVENTION

The present invention provides novel synthetic therapeutic compounds.These compounds are designed to enhance binding to MHC molecules and toenhance immunoregulatory properties relative to their naturalcounterparts. The synthetic compounds of the invention are useful tomodulate an immune response to the synthetic and naturally occurringcompounds.

Further provided are polynucleotides encoding the compounds of theinvention, gene delivery vehicles comprising these polynucleotides andhost cells comprising these polynucleotides.

In addition, the invention provides methods for inducing an immuneresponse in a subject by delivering the compounds and compositions ofthe invention, and delivering these in the context of an MHC molecule.

The compounds of the invention are also useful to generate antibodiesthat specifically recognize and bind to these molecules. Theseantibodies are further useful for immunotherapy when administered to asubject.

The invention also provides immune effector cells raised in vivo or invitro in the presence and at the expense of an antigen presenting cellthat presents the peptide compositions of the invention in the contextof an MHC molecule and a method of adoptive immunotherapy comprisingadministering an effective amount of these immune effector cells to asubject.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1. The complete nucleotide sequence of a cDNA encoding the CMVantigen pp65.

SEQ ID NO:2. The amino acid sequence of the native CMV antigen pp65. Thecompounds of the invention are variations based on native pp65 peptide495–503.

SEQ ID NO:3. The amino acid sequence of compound 1.

SEQ ID NO:4. The polynucleotide sequence encoding compound 1.

SEQ ID NO:5. The amino acid sequence of compound 2.

SEQ ID NO:6. The polynucleotide sequence encoding compound 2.

SEQ ID NO:7. The amino acid sequence of compound 3.

SEQ ID NO:8. The polynucleotide sequence encoding compound 3.

SEQ ID NO:9. The amino acid sequence of compound 4.

SEQ ID NO:10. The polynucleotide sequence encoding compound 4.

SEQ ID NO:11. The amino acid sequence of compound 5.

SEQ ID NO:12. The polynucleotide sequence encoding compound 5.

SEQ ID NO:13. The amino acid sequence of compound 6.

SEQ ID NO:14. The polynucleotide sequence encoding compound 6.

SEQ ID NO:15. The amino acid sequence of the native pp65 antigenicpeptide 495–503.

MODES OF CARRYING OUT THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. These methods are described in thefollowing publications. See, e.g., Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2^(nd) edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al. eds. (1987)); the series METHODS INENZYMOLOGY (Academic Press, Inc.); PCR: A PRACTICAL APPROACH (M.MacPherson et al. IRL Press at Oxford University Press (1991)); PCR 2: APRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.(1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow and Lane eds. (1988));and ANIMAL CELL CULTURE (R. I. Freshney ed. (1987)).

Definitions

As used herein, certain terms may have the following defined meanings.

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

A “native” or “natural” antigen is a polypeptide, protein or a fragmentwhich contains an epitope, which has been isolated from a naturalbiological source, and which can specifically bind to an antigenreceptor, in particular a T cell antigen receptor (TCR), in a subject.

The term “antigen” is well understood in the art and includes substanceswhich are immunogenic, i.e., immunogens, as well as substances whichinduce immunological unresponsiveness, or anergy, i.e., anergens.

An “altered antigen” is one having a primary sequence that is differentfrom that of the corresponding wild-type antigen. Altered antigens canbe made by synthetic or recombinant methods and include, but are notlimited to, antigenic peptides that are differentially modified duringor after translation, e.g., by phosphorylation, glycosylation,cross-linking, acylation, proteolytic cleavage, linkage to an antibodymolecule, membrane molecule or other ligand. (Ferguson et al. (1988)Ann. Rev. Biochem. 57:285–320). A synthetic or altered antigen of theinvention is intended to bind to the same TCR as the natural epitope.

A “self-antigen” also referred to herein as a native or wild-typeantigen is an antigenic peptide that induces little or no immuneresponse in the subject due to self-tolerance to the antigen. An exampleof a self-antigen is the melanoma specific antigen gp100.

The term “tumor associated antigen” or “TAA” refers to an antigen thatis associated with or specific to a tumor. Examples of known TAAsinclude gp100, MART and MAGE.

The terms “major histocompatibility complex” or “MHC” refers to acomplex of genes encoding cell-surface molecules that are required forantigen presentation to T cells and for rapid graft rejection. Inhumans, the MHC is also known as the “human leukocyte antigen” or “HLA”complex. The proteins encoded by the MHC are known as “MHC molecules”and are classified into class I and class II MHC molecules. Class I MHCincludes membrane heterodimeric proteins made up of an α chain encodedin the MHC noncovalently linked with the β2-microglobulin. Class I MHCmolecules are expressed by nearly all nucleated cells and have beenshown to function in antigen presentation to CD8⁺ T cells. Class Imolecules include HLA-A, B, and C in humans. Class II MHC molecules alsoinclude membrane heterodimeric proteins consisting of noncovalentlyassociated α and β chains. Class II MHC molecules are known to functionin CD4⁺ T cells and, in humans, include HLA-DP, -DQ, and DR. In apreferred embodiment, invention compositions and ligands can complexwith MHC molecules of any HLA type. Those of skill in the art arefamiliar with the serotypes and genotypes of the HLA. See:http://bimas.dcrt.nih.gov/cgi-bin/molbio/hla_coefficient_viewing_page.Rammensee H. G., Bachmann J., and Stevanovic S. MHC Ligands and PeptideMotifs (1997) Chapman & Hall Publishers; Schreuder G. M. Th. et al. TheHLA dictionary (1999) Tissue Antigens 54:409–437.

The term “antigen-presenting matrix”, as used herein, intends a moleculeor molecules which can present antigen in such a way that the antigencan be bound by a T-cell antigen receptor on the surface of a T cell. Anantigen-presenting matrix can be on the surface of an antigen-presentingcell (APC), on a vesicle preparation of an APC, or can be in the form ofa synthetic matrix on a solid support such as a bead or a plate. Anexample of a synthetic antigen-presenting matrix is purified MHC class Imolecules complexed to β2-microglobulin, multimers of such purified MHCclass I molecules, purified MHC Class II molecules, or functionalportions thereof, attached to a solid support.

The term “antigen presenting cells (APC)” refers to a class of cellscapable of presenting one or more antigens in the form of antigen-MHCcomplex recognizable by specific effector cells of the immune system,and thereby inducing an effective cellular immune response against theantigen or antigens being presented. While many types of cells may becapable of presenting antigens on their cell surface for T-cellrecognition, only professional APCs have the capacity to presentantigens in an efficient amount and further to activate T-cells forcytotoxic T-lymphocyte (CTL) responses. APCs can be intact whole cellssuch as macrophages, B-cells and dendritic cells; or other molecules,naturally occurring or synthetic, such as purified MHC class I moleculescomplexed to β2-microglobulin.

The term “dendritic cells (DC)” refers to a diverse population ofmorphologically similar cell types found in a variety of lymphoid andnon-lymphoid tissues (Steinman (1991) Ann. Rev. Immunol. 9:271–296).Dendritic cells constitute the most potent and preferred APCs in theorganism. A subset, if not all, of dendritic cells are derived from bonemarrow progenitor cells, circulate in small numbers in the peripheralblood and appear either as immature Langerhans' cells or terminallydifferentiated mature cells. While the dendritic cells can bedifferentiated from monocytes, they possess distinct phenotypes. Forexample, a particular differentiating marker, CD 14 antigen, is notfound in dendritic cells but is possessed by monocytes. Also, maturedendritic cells are not phagocytic, whereas the monocytes are stronglyphagocytosing cells. It has been shown that DCs provide all the signalsnecessary for T cell activation and proliferation.

The term “antigen presenting cell recruitment factors” or “APCrecruitment factors” include both intact, whole cells as well as othermolecules that are capable of recruiting antigen presenting cells.Examples of suitable APC recruitment factors include molecules such asinterleukin 4 (IL4), granulocyte macrophage colony stimulating factor(GM-CSF), Sepragel and macrophage inflammatory protein 3 alpha (MIP3α).These are available from Immunex, Schering-Plough and R&D Systems(Minneapolis, Minn.). They also can be recombinantly produced using themethods disclosed in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M.Ausubel et al., eds. (1987)). Peptides, proteins and compounds havingthe same biological activity as the above-noted factors are includedwithin the scope of this invention.

The term “immune effector cells” refers to cells capable of binding anantigen and which mediate an immune response. These cells include, butare not limited to, T cells, B cells, monocytes, macrophages, NK cellsand cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones,and CTLs from tumor, inflammatory, or other infiltrates. Certaindiseased tissue expresses specific antigens and CTLs specific for theseantigens have been identified. For example, approximately 80% ofmelanomas express the antigen known as gp100.

The term “immune effector molecule” as used herein, refers to moleculescapable of antigen-specific binding, and includes antibodies, T cellantigen receptors, and MHC Class I and Class II molecules.

A “naïve” immune effector cell is an immune effector cell that has neverbeen exposed to an antigen capable of activating that cell. Activationof naïve immune effector cells requires both recognition of thepeptide:MHC complex and the simultaneous delivery of a costimulatorysignal by a professional APC in order to proliferate and differentiateinto antigen-specific armed effector T cells.

“Immune response” broadly refers to the antigen-specific responses oflymphocytes to foreign substances. Any substance that can elicit animmune response is said to be “immunogenic” and is referred to as an“immunogen”. All immunogens are antigens, however, not all antigens areimmunogenic. An immune response of this invention can be humoral (viaantibody activity) or cell-mediated (via T cell activation).

The term “ligand” as used herein refers to any molecule that binds to aspecific site on another molecule. In other words, the ligand confersthe specificity of the protein in a reaction with an immune effectorcell. It is the ligand site within the protein that combines directlywith the complementary binding site on the immune effector cell.

In a preferred embodiment, a ligand of the invention binds to anantigenic determinant or epitope on an immune effector cell, such as anantibody or a T cell receptor (TCR). A ligand may be an antigen,peptide, protein or epitope of the invention.

Invention ligands may bind to a receptor on an antibody. In oneembodiment, the ligand of the invention is about 4 to about 8 aminoacids in length.

Invention ligands may bind to a receptor on an MHC class I molecule. Inone embodiment, the ligand of the invention is about 7 to about 11 aminoacids in length.

Invention ligands may bind to a receptor on an MHC class II molecule. Inone embodiment, the ligand of the invention is about 10 to about 20amino acids long.

As used herein, the term “educated, antigen-specific immune effectorcell”, is an immune effector cell as defined above, which has previouslyencountered an antigen. In contrast with its naïve counterpart,activation of an educated, antigen-specific immune effector cell doesnot require a costimulatory signal. Recognition of the peptide:MHCcomplex is sufficient.

“Activated”, when used in reference to a T cell, implies that the cellis no longer in Go phase, and begins to produce one or more ofcytotoxins, cytokines, and other related membrane-associated proteinscharacteristic of the cell type (e.g., CD8⁺ or CD4⁺), is capable ofrecognizing and binding any target cell that displays the particularantigen on its surface, and releasing its effector molecules.

In the context of the present invention, the term “recognized” intendsthat a composition of the invention, comprising one or more ligands, isrecognized and bound by an immune effector cell wherein such bindinginitiates an effective immune response. Assays for determining whether aligand is recognized by an immune effector cell are known in the art andare described herein.

The term “preferentially recognized” intends that the specificity of acomposition or ligand of the invention is restricted to immune effectorcells that recognize and bind the native ligand.

The term “cross-reactive” is used to describe compounds of the inventionwhich are functionally overlapping. More particularly, the immunogenicproperties of a native ligand and/or immune effector cells activatedthereby are shared to a certain extent by the altered ligand such thatthe altered ligand is “cross-reactive” with the native ligand and/or theimmune effector cells activated thereby. For purposes of this invention,cross-reactivity is manifested at multiple levels: (i) at the ligandlevel, e.g., the altered ligands can bind the TCR of and activate nativeligand CTLs; (ii) at the T cell level, i.e., altered ligands of theinvention bind the TCR of and activate a population of T cells (distinctfrom the population of native ligand CTLs) which can effectively targetand lyse cells displaying the native ligand; and (iii) at the antibodylevel, e.g., “anti”-altered ligand antibodies can detect, recognize andbind the native ligand and initiate effector mechanisms in an immuneresponse which ultimately result in elimination of the native ligandfrom the host.

As used herein, the term “inducing an immune response in a subject” is aterm well understood in the art and intends that an increase of at leastabout 2-fold, more preferably at least about 5-fold, more preferably atleast about 10-fold, more preferably at least about 100-fold, even morepreferably at least about 500-fold, even more preferably at least about1000-fold or more in an immune response to an antigen (or epitope) canbe detected or measured, after introducing the antigen (or epitope) intothe subject, relative to the immune response (if any) beforeintroduction of the antigen (or epitope) into the subject. An immuneresponse to an antigen (or epitope), includes, but is not limited to,production of an antigen-specific (or epitope-specific) antibody, andproduction of an immune cell expressing on its surface a molecule whichspecifically binds to an antigen (or epitope). Methods of determiningwhether an immune response to a given antigen (or epitope) has beeninduced are well known in the art. For example, antigen-specificantibody can be detected using any of a variety of immunoassays known inthe art, including, but not limited to, ELISA, wherein, for example,binding of an antibody in a sample to an immobilized antigen (orepitope) is detected with a detectably-labeled second antibody (e.g.,enzyme-labeled mouse anti-human Ig antibody).

“Co-stimulatory molecules” are involved in the interaction betweenreceptor-ligand pairs expressed on the surface of antigen presentingcells and T cells. Research accumulated over the past several years hasdemonstrated convincingly that resting T cells require at least twosignals for induction of cytokine gene expression and proliferation(Schwartz R. H. (1990) Science 248:1349–1356 and Jenkins M. K. (1992)Immunol. Today 13:69–73). One signal, the one that confers specificity,can be produced by interaction of the TCR/CD3 complex with anappropriate MHC/peptide complex. The second signal is not antigenspecific and is termed the “co-stimulatory” signal. This signal wasoriginally defined as an activity provided by bone-marrow-derivedaccessory cells such as macrophages and dendritic cells, the so called“professional” APCs. Several molecules have been shown to enhanceco-stimulatory activity. These are heat stable antigen (HSA) (Liu Y. etal. (1992) J. Exp. Med. 175:437–445), chondroitin sulfate-modified MHCinvariant chain (Ii-CS) (Naujokas M. F. et al. (1993) Cell 74:257–268),intracellular adhesion molecule 1 (ICAM-1) (Van Seventer G. A. (1990) J.Immunol. 144:4579–4586), B7-1, and B7-2/B70 (Schwartz R. H. (1992) Cell71:1065–1068). These molecules each appear to assist co-stimulation byinteracting with their cognate ligands on the T cells. Co-stimulatorymolecules mediate co-stimulatory signal(s), which are necessary, undernormal physiological conditions, to achieve full activation of naïve Tcells. One exemplary receptor-ligand pair is the B7 co-stimulatorymolecule on the surface of APCs and its counter-receptor CD28 or CTLA-4on T cells (Freeman et al. (1993) Science 262:909–911; Young et al.(1992) J. Clin. Invest. 90:229 and Nabavi et al. (1992) Nature360:266–268). Other important co-stimulatory molecules are CD40, CD54,CD80, and CD86. The term “co-stimulatory molecule” encompasses anysingle molecule or combination of molecules which, when acting togetherwith a peptide/MHC complex bound by a TCR on the surface of a T cell,provides a co-stimulatory effect which achieves activation of the T cellthat binds the peptide. The term thus encompasses B7, or otherco-stimulatory molecule(s) on an antigen-presenting matrix such as anAPC, fragments thereof (alone, complexed with another molecule(s), or aspart of a fusion protein) which, together with peptide/MHC complex,binds to a cognate ligand and results in activation of the T cell whenthe TCR on the surface of the T cell specifically binds the peptide.Co-stimulatory molecules are commercially available from a variety ofsources, including, for example, Beckman Coulter, Inc. (Fullerton,Calif.). It is intended, although not always explicitly stated, thatmolecules having similar biological activity as wild-type or purifiedco-stimulatory molecules (e.g., recombinantly produced or muteinsthereof) are intended to be used within the spirit and scope of theinvention.

As used herein, “solid phase support” or “solid support”, usedinterchangeably, is not limited to a specific type of support. Rather alarge number of supports are available and are known to one of ordinaryskill in the art. Solid phase supports include silica gels, resins,derivatized plastic films, glass beads, cotton, plastic beads, aluminagels. As used herein, “solid support” also includes syntheticantigen-presenting matrices, cells, and liposomes. A suitable solidphase support may be selected on the basis of desired end use andsuitability for various protocols. For example, for peptide synthesis,solid phase support may refer to resins such as polystyrene (e.g.,PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.),POLYHIPE® resin (obtained from Aminotech, Canada), polyamide resin(obtained from Peninsula Laboratories), polystyrene resin grafted withpolyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) orpolydimethylacrylamide resin (obtained from Milligen/Biosearch, Calif.).

The term “immunomodulatory agent”, as used herein, is a molecule, amacromolecular complex, or a cell that modulates an immune response andencompasses a synthetic antigenic peptide of the invention alone or inany of a variety of formulations described herein; a polypeptidecomprising a synthetic antigenic peptide of the invention; apolynucleotide encoding a peptide or polypeptide of the invention; asynthetic antigenic peptide of the invention bound to a Class I or aClass II MHC molecule on an antigen-presenting matrix, including an APCand a synthetic antigen-presenting matrix (in the presence or absence ofco-stimulatory molecule(s)); a synthetic antigenic peptide of theinvention covalently or non-covalently complexed to another molecule(s)or macromolecular structure; and an educated, antigen-specific immuneeffector cell which is specific for a peptide of the invention.

The term “modulate an immune response” includes inducing (increasing,eliciting) an immune response; and reducing (suppressing) an immuneresponse. An immunomodulatory method (or protocol) is one that modulatesan immune response in a subject.

As used herein, the term “cytokine” refers to any one of the numerousfactors that exert a variety of effects on cells, for example, inducinggrowth or proliferation. Non-limiting examples of cytokines which may beused alone or in combination in the practice of the present inventioninclude, interleukin 2 (IL-2), stem cell factor (SCF), interleukin 3(IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocytemacrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha(IL-1I), interleukin 11 (IL-11), MIP-11, leukemia inhibitory factor(LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The presentinvention also includes culture conditions in which one or more cytokineis specifically excluded from the medium. Cytokines are commerciallyavailable from several vendors such as, for example, Genzyme(Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen(Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.) and Immunex(Seattle, Wash.). It is intended, although not always explicitly stated,that molecules having similar biological activity as wild-type orpurified cytokines (e.g., recombinantly produced or muteins thereof) areintended to be used within the spirit and scope of the invention.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example,single-stranded, double-stranded and triple helical molecules, a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A nucleic acid molecule may alsocomprise modified nucleic acid molecules.

The term “peptide” is used in its broadest sense to refer to a compoundof two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g. ester, ether,etc. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D or L optical isomers, and amino acid analogs and peptidomimetics.A peptide of three or more amino acids is commonly called anoligopeptide if the peptide chain is short. If the peptide chain islong, the peptide is commonly called a polypeptide or a protein.

The term “genetically modified” means containing and/or expressing aforeign gene or nucleic acid sequence which in turn, modifies thegenotype or phenotype of the cell or its progeny. In other words, itrefers to any addition, deletion or disruption to a cell's endogenousnucleotides.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA, if an appropriateeukaryotic host is selected. Regulatory elements required for expressioninclude promoter sequences to bind RNA polymerase and transcriptioninitiation sequences for ribosome binding. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgamo sequence and the start codonAUG (Sambrook et al. (1989) supra). Similarly, a eukaryotic expressionvector includes a heterologous or homologous promoter for RNA polymeraseII, a downstream polyadenylation signal, the start codon AUG, and atermination codon for detachment of the ribosome. Such vectors can beobtained commercially or assembled by the sequences described in methodswell known in the art, for example, the methods described below forconstructing vectors in general.

“Under transcriptional control” is a term well understood in the art andindicates that transcription of a polynucleotide sequence, usually a DNAsequence, depends on its being operatively linked to an element whichcontributes to the initiation of, or promotes, transcription.“Operatively linked” refers to a juxtaposition wherein the elements arein an arrangement allowing them to function.

A “gene delivery vehicle” is defined as any molecule that can carryinserted polynucleotides into a host cell. Examples of gene deliveryvehicles are liposomes, biocompatible polymers, including naturalpolymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, or viruses, such as baculovirus, adenovirus andretrovirus, bacteriophage, cosmid, plasmid, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

“Gene delivery,” “gene transfer,” and the like as used herein, are termsreferring to the introduction of an exogenous polynucleotide (sometimesreferred to as a “transgene”) into a host cell, irrespective of themethod used for the introduction. Such methods include a variety ofwell-known techniques such as vector-mediated gene transfer (by, e.g.,viral infection/transfection, or various other protein-based orlipid-based gene delivery complexes) as well as techniques facilitatingthe delivery of “naked” polynucleotides (such as electroporation, “genegun” delivery and various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors, alphavirus vectors and the like. Alphavirus vectors, such asSemliki Forest virus-based vectors and Sindbis virus-based vectors, havealso been developed for use in gene therapy and immunotherapy. See,Schlesinger and Dubensky (1999) Curr Opin Biotechnol. 10(5):434–439 andYing et al. (1999) Nat. Med. 5(7):823–827. In aspects where genetransfer is mediated by a retroviral vector, a vector construct refersto the polynucleotide comprising the retroviral genome or part thereof,and a therapeutic gene. As used herein, “retroviral mediated genetransfer” or “retroviral transduction” carries the same meaning andrefers to the process by which a gene or nucleic acid sequences arestably transferred into the host cell by virtue of the virus enteringthe cell and integrating its genome into the host cell genome. The viruscan enter the host cell via its normal mechanism of infection or bemodified such that it binds to a different host cell surface receptor orligand to enter the cell. As used herein, retroviral vector refers to aviral particle capable of introducing exogenous nucleic acid into a cellthrough a viral or viral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes.See, e.g., WO 95/27071. Ads are easy to grow and do not requireintegration into the host cell genome. Recombinant Ad-derived vectors,particularly those that reduce the potential for recombination andgeneration of wild-type virus, have also been constructed. See, WO95/00655 and WO 95/11984. Wild-type AAV has high infectivity andspecificity integrating into the host cell's genome. See, Hermonat andMuzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466–6470 and Lebkowski etal. (1988) Mol. Cell. Biol. 8:3988–3996.

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include several non-viral vectors, includingDNA/liposome complexes, and targeted viral protein-DNA complexes.Liposomes that also comprise a targeting antibody or fragment thereofcan be used in the methods of this invention. To enhance delivery to acell, the nucleic acid or proteins of this invention can be conjugatedto antibodies or binding fragments thereof which bind cell surfaceantigens, e.g., TCR, CD3 or CD4.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10×SSC; formamide concentrationsof about 0% to about 25%; and wash solutions of about 6×SSC. Examples ofmoderate hybridization conditions include: incubation temperatures ofabout 40° C. to about 50° C.; buffer concentrations of about 9×SSC toabout 2×SSC; formamide concentrations of about 30% to about 50%; andwash solutions of about 5×SSC to about 2×SSC. Examples of highstringency conditions include: incubation temperatures of about 55° C.to about 68° C.; buffer concentrations of about 1×SSC to about 0.1×SSC;formamide concentrations of about 55% to about 75%; and wash solutionsof about 1×SSC, 0.1×SSC, or deionized water. In general, hybridizationincubation times are from 5 minutes to 24 hours, with 1, 2, or morewashing steps, and wash incubation times are about 1, 2, or 15 minutes.SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood thatequivalents of SSC using other buffer systems can be employed.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 80%, 85%,90%, or 95%) of“sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. This alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18, Table 7.7.1. Preferably, default parameters are used foralignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address:http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

“In vivo” gene delivery, gene transfer, gene therapy and the like asused herein, are terms referring to the introduction of a vectorcomprising an exogenous polynucleotide directly into the body of anorganism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced to a cell of such organism in vivo.

The term “isolated” means separated from constituents, cellular andotherwise, in which the polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, are normally associated with in nature.For example, with respect to a polynucleotide, an isolatedpolynucleotide is one that is separated from the 5′ and 3′ sequenceswith which it is normally associated in the chromosome. As is apparentto those of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, antibody, or fragments thereof, does notrequire “isolation” to distinguish it from its naturally occurringcounterpart. In addition, a “concentrated”, “separated” or “diluted”polynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, is distinguishable from its naturally occurring counterpart inthat the concentration or number of molecules per volume is greater than“concentrated” or less than “separated” than that of its naturallyoccurring counterpart. A polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, which differs from the naturallyoccurring counterpart in its primary sequence or for example, by itsglycosylation pattern, need not be present in its isolated form since itis distinguishable from its naturally occurring counterpart by itsprimary sequence, or alternatively, by another characteristic such asglycosylation pattern. Although not explicitly stated for each of theinventions disclosed herein, it is to be understood that all of theabove embodiments for each of the compositions disclosed below and underthe appropriate conditions, are provided by this invention. Thus, anon-naturally occurring polynucleotide is provided as a separateembodiment from the isolated naturally occurring polynucleotide. Aprotein produced in a bacterial cell is provided as a separateembodiment from the naturally occurring protein isolated from aeukaryotic cell in which it is produced in nature.

“Host cell,” “target cell” or “recipient cell” are intended to includeany individual cell or cell culture which can be or have been recipientsfor vectors or the incorporation of exogenous nucleic acid molecules,polynucleotides and/or proteins. It also is intended to include progenyof a single cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic or total DNA complement) to theoriginal parent cell due to natural, accidental, or deliberate mutation.The cells may be prokaryotic or eukaryotic, and include but are notlimited to bacterial cells, yeast cells, animal cells, and mammaliancells, e.g., murine, rat, simian or human.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative”. Forexample, where the purpose of the experiment is to determine acorrelation of an altered expression level of a gene with a particulartype of cancer, it is generally preferable to use a positive control (asubject or a sample from a subject, carrying such alteration andexhibiting syndromes characteristic of that disease), and a negativecontrol (a subject or a sample from a subject lacking the alteredexpression and clinical syndrome of that disease).

The terms “cancer,” “neoplasm,” and “tumor,” used interchangeably and ineither the singular or plural form, refer to cells that have undergone amalignant transformation that makes them pathological to the hostorganism. Primary cancer cells (that is, cells obtained from near thesite of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical orimmunologic findings alone may be insufficient to meet this definition.

“Suppressing” tumor growth indicates a growth state that is curtailedcompared to growth without contact with educated, antigen-specificimmune effector cells described herein. Tumor cell growth can beassessed by any means known in the art, including, but not limited to,measuring tumor size, determining whether tumor cells are proliferatingusing a ³H-thymidine incorporation assay, or counting tumor cells.“Suppressing” tumor cell growth means any or all of the followingstates: slowing, delaying, and “suppressing” tumor growth indicates agrowth state that is curtailed when stopping tumor growth, as well astumor shrinkage.

The term “culturing” refers to the in vitro propagation of cells ororganisms on or in media of various kinds. It is understood that thedescendants of a cell grown in culture may not be completely identical(morphologically, genetically, or phenotypically) to the parent cell. By“expanded” is meant any proliferation or division of cells.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin REMINGTON'SPHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

The present invention provides compounds having the followingstructures:

The present invention also provides compositions that exhibit enhancingbinding to MHC molecules and are cross-reactive with and useful formodulating immune responses to the cognate native ligands and theircorresponding native proteins.

This invention further provides compositions which are useful ascomponents of anti-cancer vaccines and to expand immune effector cellsthat are specific for cancers characterized by expression of the CMVantigen.

In one embodiment, the altered ligands of the invention have comparableaffinity for MHC binding as the native ligand. It has been demonstratedthat peptide:MHC class I binding properties correlate withimmunogenicity (Sette A. et al. (1994) J. Immunol. 153(12):5586–5592;van der Burg S. H. et al. (1996) J. Immunol. 156:3308–3314). In apreferred embodiment, altered ligands of the invention bind to a TCRwith a higher affinity than of that the “natural” ligand. Comparativebinding of the native and altered ligands of the invention to an MHCclass I molecule can be measured by methods that are known in the artand include, but are not limited to, calculating the affinity based onan algorithm (see, for example, Parker et al. (1992) J. Immunol.149:3580–3587) and experimentally determining binding affinity (see, forexample, Tan et al. (1997) J. Immunol. Meth. 209(1):25–36). For example,the relative binding of a peptide to a class I molecule can be measuredon the basis of binding of a radiolabeled standard peptide todetergent-solubilized MHC molecules, using various concentrations oftest peptides (e.g., ranging from 100 mM to 1 nM). MHC class I heavychain and β2-microglobulin are coincubated with a fixed concentration(e.g., 5 nM) radiolabeled standard (control) peptide and variousconcentrations of a test peptide for a suitable period of time (e.g., 2hours to 72 hours) at room temperature in the presence of a mixture ofprotease inhibitors. A control tube contains standard peptide and MHCmolecules, but no test peptide. The percent MHC-bound radioactivity isdetermined by gel filtration. The IC₅₀ (concentration of test peptidewhich results in 50% inhibition of binding of control peptide) iscalculated for each peptide. Additional methods for determining bindingaffinity to a TCR are known in the art and include, but are not limitedto, those described in al-Ramadi et al. (1992) J. Immunol.155(2):662–673; and Zuegel et al. (1998) J. Immunol. 161(4):1705–1709.

In another embodiment, the altered ligands of the invention elicitcomparable antigen-specific T cell activation relative to their nativeligand counterpart. In a preferred embodiment, altered ligands of theinvention elicit a stronger antigen-specific T cell activation relativeto their native ligand counterpart. Methods for determiningimmunogenicity of invention ligands are known in the art and are furtherdescribed herein.

In one embodiment, compositions of the invention comprise two or moreimmunogenic ligands of the invention. In one aspect, such compositionsmay comprise two or more copies of a single ligand. In another aspect,such compositions may comprise two or more ligands, wherein each ligandof said two or more ligands is distinct from all other ligands in saidcomposition. In one embodiment, the two or more immunogenic ligands arecovalently linked.

The present invention also provides novel synthetic antigenic peptidesdesigned for enhancing binding to MHC molecules and useful formodulating immune responses to the synthetic peptide epitope and thecorresponding native peptides from which they are derived. The syntheticantigenic peptide epitope sequences of the present invention differ fromtheir natural counterparts in that they contain alterations in aminoacid sequence, relative to the native sequence, in the MHC Class Ibinding domain which is designed to confer tighter binding to the MHC.They further contain mutations in the putative T cell receptor-bindingdomain designed to increase affinity for the T cell antigen receptor.These differences from the native sequence are designed to conferadvantages in the methods of the present invention over the nativesequence, in that the synthetic antigenic peptide epitopes of theinvention will have enhanced immunomodulatory properties.

This invention provides novel, synthetic antigenic peptide sequences,which are useful as components of anti-viral vaccines and to expandimmune effector cells that are specific for viral infectionscharacterized by expression of the CMV antigen pp65. The peptides,FLLPMIATV (SEQ ID NO:3), FLLWDWPFV (SEQ ID NO:5), FLFTRFMRV (SEQ IDNO:7), FLPHPGWLV (SEQ ID NO:9), FLIRLTPPV (SEQ ID NO:11), and FLDFSFWFV(SEQ ID NO:13) differ from the natural CMV epitope NLVPMVATV (SEQ IDNO:2) in two ways: (1) they contain mutations in the putative HLA-A2binding domain (specifically amino acid residue 1) conferring tighterbinding to the MHC, and (2) they contain mutations in the putative Tcell receptor-binding domain (amino acid residues 3–8) resulting in anincreased avidity for the T cell receptor.

Binding of synthetic antigenic peptide of the invention to an MHC ClassI molecule can be measured by methods that are known in the art andinclude, but are not limited to, calculating the affinity based on analgorithm (see, for example, Parker et al. (1992) J. Immunol.149:3580–3587); and experimentally determining binding affinity (see,for example, Tan et al. (1997) J. Immunol. Meth. 209(1):25–36). Forexample, the relative binding of a peptide to a Class I molecule can bemeasured on the basis of binding of a radiolabeled standard peptide todetergent-solubilized MHC molecules, using various concentrations oftest peptides (e.g., ranging from 100 mM to 1 nM). MHC Class I heavychain and β2-microglobulin are coincubated with a fixed concentration(e.g., 5 nM) radiolabeled standard (control) peptide and variousconcentrations of a test peptide for a suitable period of time (e.g., 2hours to 72 hours) at room temperature in the presence of a mixture ofprotease inhibitors. A control tube contains standard peptide and MHCmolecules, but no test peptide. The percent MHC-bound radioactivity isdetermined by gel filtration. The IC₅₀ (concentration of test peptidewhich results in 50% inhibition of binding of control peptide) iscalculated for each peptide.

Synthetic peptides of the invention are designed to bind to a TCR with ahigher affinity than of that the “natural” sequence. Methods fordetermining binding affinity to a TCR are known in the art and include,but are not limited to, those described in al-Ramadi et al. (1992) J.Immunol. 155(2):662–673; and Zuegel et al. (1998) J. Immunol.161(4):1705–1709.

Further encompassed by the term “synthetic antigenic peptide” aremultimers (concatemers) of a synthetic antigenic peptide of theinvention, optionally including intervening amino acid sequences as wellas polypeptides comprising the sequences FLLPMIATV (SEQ ID NO:3),FLLWDWPFV (SEQ ID NO:5), FLFTRFMRV (SEQ ID NO:7), FLPHPGWLV (SEQ IDNO:9), FLIRLTPPV (SEQ ID NO:11) and FLDFSFWFV (SEQ ID NO:13). Theinvention also provides polypeptides comprising these sequences whereinthe polypeptides are preferentially recognized by CMV antigen pp65specific cytotoxic T lymphocytes.

Polypeptides comprising the peptide sequences of the invention can beprepared by altering the sequence of polynucleotides that encode thenative CMV antigen pp65 polypeptide sequence. This is accomplished bymethods of recombinant DNA technology well know to those skilled in theart. For example, site directed mutagenesis can be performed onrecombinant polynucleotides encoding the native CMV antigen pp65sequence to introduce changes in the polynucleotide sequence so that thealtered polynucleotide encodes the peptides of the invention.

The proteins and polypeptides of this invention can be obtained bychemical synthesis using a commercially available automated peptidesynthesizer such as those manufactured by Perkin Elmer/AppliedBiosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. Thesynthesized protein or polypeptide can be precipitated and furtherpurified, for example by high performance liquid chromatography (HPLC).Accordingly, this invention also provides a process for chemicallysynthesizing the proteins of this invention by providing the sequence ofthe protein and reagents, such as amino acids and enzymes and linkingtogether the amino acids in the proper orientation and linear sequence.

Alternatively, the proteins and polypeptides can be obtained bywell-known recombinant methods as described herein using the host celland vector systems described below.

Peptide Analogues

It is well know to those skilled in the art that modifications can bemade to the peptides of the invention to provide them with alteredproperties. As used herein the term “amino acid” refers to eithernatural and/or unnatural or synthetic amino acids, including glycine andboth the D or L optical isomers, and amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlycalled an oligopeptide if the peptide chain is short. If the peptidechain is long, the peptide is commonly called a polypeptide or aprotein.

Peptides of the invention can be modified to include unnatural aminoacids. Thus, the peptides may comprise D-amino acids, a combination ofD- and L-amino acids, and various “designer” amino acids (e.g., β-methylamino acids, C-α-methyl amino acids, and N-α-methyl amino acids, etc.)to convey special properties to peptides. Additionally, by assigningspecific amino acids at specific coupling steps, peptides with α-helicesβ turns, β sheets, γ-turns, and cyclic peptides can be generated.Generally, it is believed that α-helical secondary structure or randomsecondary structure is preferred.

In a further embodiment, subunits of peptides that confer usefulchemical and structural properties will be chosen. For example, peptidescomprising D-amino acids will be resistant to L-amino acid-specificproteases in vivo. Modified compounds with D-amino acids may besynthesized with the amino acids aligned in reverse order to produce thepeptides of the invention as retro-inverso peptides. In addition, thepresent invention envisions preparing peptides that have better definedstructural properties, and the use of peptidomimetics, andpeptidomimetic bonds, such as ester bonds, to prepare peptides withnovel properties. In another embodiment, a peptide may be generated thatincorporates a reduced peptide bond, i.e., R₁—CH₂NH—R₂, where R₁, and R₂are amino acid residues or sequences. A reduced peptide bond may beintroduced as a dipeptide subunit. Such a molecule would be resistant topeptide bond hydrolysis, e.g., protease activity. Such molecules wouldprovide ligands with unique function and activity, such as extendedhalf-lives in vivo due to resistance to metabolic breakdown, or proteaseactivity. Furthermore, it is well known that in certain systemsconstrained peptides show enhanced functional activity (Hruby (1982)Life Sciences 31:189–199 and Hruby et al. (1990) Biochem J.268:249–262); the present invention provides a method to produce aconstrained peptide that incorporates random sequences at all otherpositions.

Non-Classical Amino Acids that Induce Conformational Constraints.

The following non classical amino acids may be incorporated in thepeptides of the invention in order to introduce particularconformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate(Kazmierski et al. (1991) J. Am. Chem. Soc. 113:2275–2283);(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine(Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41):5769–5772);2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D.Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.(1984) J. Takeda Res. Labs. 43:53–76) histidine isoquinoline carboxylicacid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131–138); andHIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep.Protein Res. 42(1):68–77) and ((1992) Acta. Cryst., Crystal Struc. Comm.48(IV):1239–1241).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834–5838);β-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett.29:5081–5082); β-turn inducing analogs (Kemp et al. (1988) TetrahedronLett. 29:5057–5060); α-helix inducing analogs (Kemp et al. (1988)Tetrahedron Lett. 29:4935–4938); γ-turn inducing analogs (Kemp et al.(1989) J. Org. Chem. 54:109:115); analogs provided by the followingreferences: Nagai and Sato (1985) Tetrahedron Lett. 26:647–650; andDiMaio et al. (1989) J. Chem. Soc. Perkin Trans. p. 1687; a Gly-Ala turnanalog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bondisostere (Jones et al. (1988) Tetrahedron Lett. 29(31):3853–3856);tetrazol (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875–5880); DTC(Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509); andanalogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323–333 andGarvey et al. (1990) J. Org. Chem. 55(3):936–940. Conformationallyrestricted mimetics of beta turns and beta bulges, and peptidescontaining them, are described in U.S. Pat. No. 5,440,013, issued Aug.8, 1995 to Kahn.

A synthetic antigenic peptide epitope of the invention can be used in avariety of formulations, which may vary depending on the intended use.

A synthetic antigenic peptide epitope of the invention can be covalentlyor non-covalently linked (complexed) to various other molecules, thenature of which may vary depending on the particular purpose. Forexample, a peptide of the invention can be covalently or non-covalentlycomplexed to a macromolecular carrier, including, but not limited to,natural and synthetic polymers, proteins, polysaccharides, polypeptides(amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids. Apeptide can be conjugated to a fatty acid, for introduction into aliposome. U.S. Pat. No. 5,837,249. A synthetic peptide of the inventioncan be complexed covalently or non-covalently with a solid support, avariety of which are known in the art. A synthetic antigenic peptideepitope of the invention can be associated with an antigen-presentingmatrix with or without co-stimulatory molecules, as described in moredetail below.

Examples of protein carriers include, but are not limited to,superantigens, serum albumin, tetanus toxoid, ovalbumin, thyroglobulin,myoglobulin, and immunoglobulin.

Peptide-protein carrier polymers may be formed using conventionalcross-linking agents such as carbodimides. Examples of carbodimides are1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC),1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

Examples of other suitable cross-linking agents are cyanogen bromide,glutaraldehyde and succinic anhydride. In general, any of a number ofhomo-bifunctional agents including a homo-bifunctional aldehyde, ahomo-bifunctional epoxide, a homo-bifunctional imido-ester, ahomo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctionalmaleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctionalhydrazide, a homo-bifunctional diazonium derivative and ahomo-bifunctional photoreactive compound may be used. Also included arehetero-bifunctional compounds, for example, compounds having anamine-reactive and a sulfhydryl-reactive group, compounds with anamine-reactive and a photoreactive group and compounds with acarbonyl-reactive and a sulfhydryl-reactive group.

Specific examples of such homo-bifunctional cross-linking agents includethe bifunctional N-hydroxysuccinimide estersdithiobis(succinimidylpropionate), disuccinimidyl suberate, anddisuccinimidyl tartarate; the bifunctional imido-esters dimethyladipimidate, dimethyl pimelimidate, and dimethyl suberimidate; thebifunctional sulfhydryl-reactive crosslinkers1,4-di-[3′-(2′-pyridyldithio) propion-amido]butane, bismaleimidohexane,and bis-N-maleimido-1, 8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as a1a′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively.

Examples of common hetero-bifunctional cross-linking agents that may beused to effect the conjugation of proteins to peptides include, but arenot limited to, SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(γ-maleimidobutyryloxy)succinimide ester), MPBH(4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-á-methyl-á-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate).

Cross-linking may be accomplished by coupling a carbonyl group to anamine group or to a hydrazide group by reductive amination.

Peptides of the invention also may be formulated as non-covalentattachment of monomers through ionic, adsorptive, or biospecificinteractions. Complexes of peptides with highly positively or negativelycharged molecules may be done through salt bridge formation under lowionic strength environments, such as in deionized water. Large complexescan be created using charged polymers such as poly-(L-glutamic acid) orpoly-(L-lysine) which contain numerous negative and positive charges,respectively. Adsorption of peptides may be done to surfaces such asmicroparticle latex beads or to other hydrophobic polymers, formingnon-covalently associated peptide-superantigen complexes effectivelymimicking cross-linked or chemically polymerized protein. Finally,peptides may be non-covalently linked through the use of biospecificinteractions between other molecules. For instance, utilization of thestrong affinity of biotin for proteins such as avidin or streptavidin ortheir derivatives could be used to form peptide complexes. Thesebiotin-binding proteins contain four binding sites that can interactwith biotin in solution or be covalently attached to another molecule.Wilchek (1988) Anal. Biochem. 171:1–32. Peptides can be modified topossess biotin groups using common biotinylation reagents such as theN-hydroxysuccinimidyl ester of D-biotin (NHS-biotin) which reacts withavailable amine groups on the protein. Biotinylated peptides then can beincubated with avidin or streptavidin to create large complexes. Themolecular mass of such polymers can be regulated through careful controlof the molar ratio of biotinylated peptide to avidin or streptavidin.

Also provided by this application are the peptides and polypeptidesdescribed herein conjugated to a detectable agent for use in thediagnostic methods. For example, detectably labeled peptides andpolypeptides can be bound to a column and used for the detection andpurification of antibodies. They also are useful as immunogens for theproduction of antibodies, as described below.

The peptides of this invention also can be combined with various liquidphase carriers, such as sterile or aqueous solutions, pharmaceuticallyacceptable carriers, suspensions and emulsions. Examples of non-aqueoussolvents include propyl ethylene glycol, polyethylene glycol andvegetable oils. When used to prepare antibodies, the carriers also caninclude an adjuvant that is useful to non-specifically augment aspecific immune response. A skilled artisan can easily determine whetheran adjuvant is required and select one. However, for the purpose ofillustration only, suitable adjuvants include, but are not limited to,Freund's Complete and Incomplete, mineral salts and polynucleotides.

This invention further provides polynucleotides (SEQ ID NOs: 4, 6, 8,10, 12, 14) encoding polypeptides comprising the sequences FLLPMIATV(SEQ ID NO:3), FLLWDWPFV (SEQ ID NO:5), FLFTRFMRV (SEQ ID NO:7),FLPHPGWLV (SEQ ID NO:9), FLIRLTPPV (SEQ ID NO:11) and FLDFSFWFV (SEQ IDNO:13), and the complements of these polynucleotides. As used herein,the term “polynucleotide” encompasses DNA, RNA and nucleic acidmimetics. In addition to the sequences shown in SEQ ID NOs: 4, 6, 8, 10,12, and 14 or their complements, this invention also provides theanti-sense polynucleotide stand, e.g., antisense RNA to these sequencesor their complements. One can obtain an antisense RNA using thesequences provided in SEQ ID NOs: 4, 6, 8, 10, 12, and 14 and themethodology described in Vander Krol, et al. (1988) BioTechniques 6:958.

The polynucleotides of this invention can be replicated using PCR. PCRtechnology is the subject matter of U.S. Pat. Nos. 4,683,195; 4,800,159;4,754,065; and 4,683,202 and described in PCR: THE POLYMERASE CHAINREACTION (Mullis et al. eds, Birkhauser Press, Boston (1994)) andreferences cited therein.

Alternatively, one of skill in the art can use the sequences providedherein and a commercial DNA synthesizer to replicate the DNA.Accordingly, this invention also provides a process for obtaining thepolynucleotides of this invention by providing the linear sequence ofthe polynucleotide, appropriate primer molecules, chemicals such asenzymes and instructions for their replication and chemicallyreplicating or linking the nucleotides in the proper orientation toobtain the polynucleotides. In a separate embodiment, thesepolynucleotides are further isolated. Still further, one of skill in theart can insert the polynucleotide into a suitable replication vector andinsert the vector into a suitable host cell (prokaryotic or eukaryotic)for replication and amplification. The DNA so amplified can be isolatedfrom the cell by methods well known to those of skill in the art. Aprocess for obtaining polynucleotides by this method is further providedherein as well as the polynucleotides so obtained.

RNA can be obtained by first inserting a DNA polynucleotide into asuitable host cell. The DNA can be inserted by any appropriate method,e.g., by the use of an appropriate gene delivery vehicle (e.g.,liposome, plasmid or vector) or by electroporation. When the cellreplicates and the DNA is transcribed into RNA; the RNA can then beisolated using methods well known to those of skill in the art, forexample, as set forth in Sambrook et al. (1989) supra. For instance,mRNA can be isolated using various lytic enzymes or chemical solutionsaccording to the procedures set forth in Sambrook, et al. (1989) supraor extracted by nucleic-acid-binding resins following the accompanyinginstructions provided by manufactures.

Polynucleotides having at least 4 contiguous nucleotides, and morepreferably at least 5 or 6 contiguous nucleotides and most preferably atleast 10 contiguous nucleotides, and exhibiting sequence complementarityor homology to SEQ ID NOs: 3, 5, 7, 9, 11, and 13 find utility ashybridization probes.

It is known in the art that a “perfectly matched” probe is not neededfor a specific hybridization. Minor changes in probe sequence achievedby substitution, deletion or insertion of a small number of bases do notaffect the hybridization specificity. In general, as much as 20%base-pair mismatch (when optimally aligned) can be tolerated.Preferably, a probe useful for detecting the aforementioned mRNA is atleast about 80% identical to the homologous region of comparable sizecontained in the previously identified sequences identified by SEQ IDNOs: 2, 4, 6, 8, 10, 12, and 14 which correspond to previouslycharacterized genes or in sequences identified in SEQ ID NOs: 2, 4, 6,8, 10, 12, and 14. More preferably, the probe is 85% identical to thecorresponding gene sequence after alignment of the homologous region;even more preferably, it exhibits 90% identity.

These probes can be used in radioassays (e.g. Southern and Northern blotanalysis) to detect or monitor various cells or tissue containing thesecells. The probes also can be attached to a solid support or an arraysuch as a chip for use in high throughput screening assays for thedetection of expression of the gene corresponding to one or morepolynucleotide(s) of this invention. Accordingly, this invention alsoprovides at least one probe as defined above of the transcriptsidentified as SEQ ID NOs: 1, 4, 6, 8, 10, 12, and 14 or the complementof one of these sequences, attached to a solid support for use in highthroughput screens.

The polynucleotides of the present invention also can serve as primersfor the detection of genes or gene transcripts that are expressed inAPC, for example, to confirm transduction of the polynucleotides intohost cells. In this context, amplification means any method employing aprimer-dependent polymerase capable of replicating a target sequencewith reasonable fidelity. Amplification may be carried out by natural orrecombinant DNA-polymerases such as T7 DNA polymerase, Klenow fragmentof E. coli DNA polymerase, and reverse transcriptase. A preferred lengthof the primer is the same as that identified for probes, above.

The invention further provides the isolated polynucleotide operativelylinked to a promoter of RNA transcription, as well as other regulatorysequences for replication and/or transient or stable expression of theDNA or RNA. As used herein, the term “operatively linked” meanspositioned in such a manner that the promoter will direct transcriptionof RNA off the DNA molecule. Examples of such promoters are SP6, T4 andT7. In certain embodiments, cell-specific promoters are used forcell-specific expression of the inserted polynucleotide. Vectors whichcontain a promoter or a promoter/enhancer, with termination codons andselectable marker sequences, as well as a cloning site into which aninserted piece of DNA can be operatively linked to that promoter arewell known in the art and commercially available. For generalmethodology and cloning strategies, see GENE EXPRESSION TECHNOLOGY(Goeddel ed., Academic Press, Inc. (1991)) and references cited thereinand VECTORS: ESSENTIAL DATA SERIES (Gacesa and Ramji, eds., John Wiley &Sons, N.Y. (1994)), which contains maps, functional properties,commercial suppliers and a reference to GenEMBL accession numbers forvarious suitable vectors. Preferably, these vectors are capable oftranscribing RNA in vitro or in vivo.

Expression vectors containing these nucleic acids are useful to obtainhost vector, systems to produce proteins and polypeptides. It is impliedthat these expression vectors must be replicable in the host organismseither as episomes or as an integral part of the chromosomal DNA.Suitable expression vectors include plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, etc.Adenoviral vectors are particularly useful for introducing genes intotissues in vivo because of their high levels of expression and efficienttransformation of cells both in vitro and in vivo. When a nucleic acidis inserted into a suitable host cell, e.g., a prokaryotic or aeukaryotic cell and the host cell replicates, the protein can berecombinantly produced. Suitable host cells will depend on the vectorand can include mammalian cells, animal cells, human cells, simiancells, insect cells, yeast cells, and bacterial cells constructed usingwell known methods. See Sambrook, et al. (1989) supra. In addition tothe use of viral vector for insertion of exogenous nucleic acid intocells, the nucleic acid can be inserted into the host cell by methodswell known in the art such as transformation for bacterial cells;transfection using calcium phosphate precipitation for mammalian cells;DEAE-dextran; electroporation; or microinjection. See Sambrook et al.(1989) supra for this methodology. Thus, this invention also provides ahost cell, e.g. a mammalian cell, an animal cell (rat or mouse), a humancell, or a prokaryotic cell such as a bacterial cell, containing apolynucleotide encoding a protein or polypeptide or antibody.

The present invention also provides delivery vehicles suitable fordelivery of a polynucleotide of the invention into cells (whether invivo, ex vivo, or in vitro). A polynucleotide of the invention can becontained within a cloning or expression vector. These vectors(especially expression vectors) can in turn be manipulated to assume anyof a number of forms which may, for example, facilitate delivery toand/or entry into a cell.

When the vectors are used for gene therapy in vivo or ex vivo, apharmaceutically acceptable vector is preferred, such as areplication-incompetent retroviral or adenoviral vector.Pharmaceutically acceptable vectors containing the nucleic acids of thisinvention can be further modified for transient or stable expression ofthe inserted polynucleotide. As used herein, the term “pharmaceuticallyacceptable vector” includes, but is not limited to, a vector or deliveryvehicle having the ability to selectively target and introduce thenucleic acid into dividing cells. An example of such a vector is a“replication-incompetent” vector defined by its inability to produceviral proteins, precluding spread of the vector in the infected hostcell. An example of a replication-incompetent retroviral vector is LNL6(Miller A. D. et al. (1989) BioTechniques 7:980–990). The methodology ofusing replication-incompetent retroviruses for retroviral-mediated genetransfer of gene markers is well established (Correll et al. (1989)Proc. Natl. Acad. Sci. USA 86:8912; Bordignon (1989) Proc. Natl. Acad.Sci. USA 86(17):6748–6752; Culver K. (1991) Proc. Natl. Acad. Sci. USA88:3155; and Rill D. R. (1992) Blood 79(10):2694–2700).

These isolated host cells containing the polynucleotides of thisinvention are useful for the recombinant replication of thepolynucleotides and for the recombinant production of peptides.Alternatively, the cells may be used to induce an immune response in asubject in the methods described herein. When the host cells are antigenpresenting cells, they can be used to expand a population of immuneeffector cells such as tumor infiltrating lymphocytes which in turn areuseful in adoptive immunotherapies.

Also provided by this invention is an antibody capable of specificallyforming a complex with the polypeptides of this invention. The term“antibody” includes polyclonal antibodies and monoclonal antibodies. Theantibodies include, but are not limited to mouse, rat, and rabbit orhuman antibodies. The antibodies are useful to identify and purifypolypeptides and APCs expressing the polypeptides.

Laboratory methods for producing polyclonal antibodies and monoclonalantibodies, as well as deducing their corresponding nucleic acidsequences, are known in the art, see Harlow and Lane (1988) supra andSambrook et al. (1989) supra. The monoclonal antibodies of thisinvention can be biologically produced by introducing protein or afragment thereof into an animal, e.g., a mouse or a rabbit. The antibodyproducing cells in the animal are isolated and fused with myeloma cellsor hetero-myeloma cells to produce hybrid cells or hybridomas.Accordingly, the hybridoma cells producing the monoclonal antibodies ofthis invention also are provided.

Thus, using the protein or fragment thereof, and well known methods, oneof skill in the art can produce and screen the hybridoma cells andantibodies of this invention for antibodies having the ability to bindthe proteins or polypeptides.

If a monoclonal antibody being tested binds with the protein orpolypeptide, then the antibody being tested and the antibodies providedby the hybridomas of this invention are equivalent. It also is possibleto determine without undue experimentation, whether an antibody has thesame specificity as the monoclonal antibody of this invention bydetermining whether the antibody being tested prevents a monoclonalantibody of this invention from binding the protein or polypeptide withwhich the monoclonal antibody is normally reactive. If the antibodybeing tested competes with the monoclonal antibody of the invention asshown by a decrease in binding by the monoclonal antibody of thisinvention, then it is likely that the two antibodies bind to the same ora closely related epitope. Alternatively, one can pre-incubate themonoclonal antibody of this invention with a protein with which it isnormally reactive, and determine if the monoclonal antibody being testedis inhibited in its ability to bind the antigen. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or a closely related, epitopic specificity as the monoclonalantibody of this invention.

The term “antibody” also is intended to include antibodies of allisotypes. Particular isotypes of a monoclonal antibody can be preparedeither directly by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass switch variants using the procedure described in Steplewski et al.(1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J.Immunol. Meth. 74:307.

This invention also provides biological active fragments of thepolyclonal and monoclonal antibodies described above. These “antibodyfragments” retain some ability to selectively bind with its antigen orimmunogen. Such antibody fragments can include, but are not limited to:

Fab,

Fab′,

F(ab′)₂,

Fv, and

SCA.

A specific example of “a biologically active antibody fragment” is a CDRregion of the antibody. Methods of making these fragments are known inthe art, see for example, Harlow and Lane (1988) supra.

The antibodies of this invention also can be modified to create chimericantibodies and humanized antibodies (Oi et al. (1986) BioTechniques4(3):214). Chimeric antibodies are those in which the various domains ofthe antibodies' heavy and light chains are coded for by DNA from morethan one species.

The isolation of other hybridomas secreting monoclonal antibodies withthe specificity of the monoclonal antibodies of the invention can alsobe accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies (Herlyn et al. (1986) Science 232:100). Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing antibodies to the epitopic determinants on a monoclonal antibodyit is possible to identify other hybridomas expressing monoclonalantibodies of the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is themirror image of the epitope bound by the first monoclonal antibody.Thus, in this instance, the anti-idiotypic monoclonal antibody could beused for immunization for production of these antibodies.

As used in this invention, the term “epitope” is meant to include anydeterminant having specific affinity for the monoclonal antibodies ofthe invention. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

The antibodies of this invention can be linked to a detectable agent orlabel. There are many different labels and methods of labeling known tothose of ordinary skill in the art.

The coupling of antibodies to low molecular weight haptens can increasethe sensitivity of the assay. The haptens can then be specificallydetected by means of a second reaction. For example, it is common to usehaptens such as biotin, which reacts avidin, or dinitropherryl,pyridoxal, and fluorescein, which can react with specific anti-haptenantibodies. See Harlow and Lane (1988) supra.

The monoclonal antibodies of the invention also can be bound to manydifferent carriers. Thus, this invention also provides compositionscontaining the antibodies and another substance, active or inert.Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

Compositions containing the antibodies, fragments thereof or cell lineswhich produce the antibodies are encompassed by this invention. Whenthese compositions are to be used pharmaceutically, they are combinedwith a pharmaceutically acceptable carrier.

In another embodiment the present invention provides a method ofinducing an immune response comprising delivering the compounds andcompositions of the invention in the context of an MHC molecule. Thus,the polypeptides of this invention can be pulsed into antigen presentingcells using the methods described herein. Antigen-presenting cells,include, but are not limited to dendritic cells (DCs),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules. The methods described belowfocus primarily on DCs which are the most potent, preferred APCs. Thesehost cells containing the polypeptides or proteins are further provided.

Isolated host cells which present the polypeptides of this invention inthe context of MHC molecules are further useful to expand and isolate apopulation of educated, antigen-specific immune effector cells. Theimmune effector cells, e.g., cytotoxic T lymphocytes, are produced byculturing naïve immune effector cells with antigen-presenting cellswhich present the polypeptides in the context of MHC molecules on thesurface of the APCs. The population can be purified using methods knownin the art, e.g., FACS analysis or ficoll gradient. The methods togenerate and culture the immune effector cells as well as thepopulations produced thereby also are the inventor's contribution andinvention. Pharmaceutical compositions comprising the cells andpharmaceutically acceptable carriers are useful in adoptiveimmunotherapy. Prior to administration in vivo, the immune effectorcells are screened in vitro for their ability to lyse CMV pp65antigen-expressing tumor cells.

In one embodiment, the immune effector cells and/or the APCs aregenetically modified. Using standard gene transfer, genes coding forco-stimulatory molecules and/or stimulatory cytokines can be insertedprior to, concurrent to or subsequent to expansion of the immuneeffector cells.

This invention also provides methods of inducing an immune response in asubject, comprising administering to the subject an effective amount ofthe polypeptides described above under the conditions that induce animmune response to the polypeptide. The polypeptides can be administeredin formulations or as polynucleotides encoding the polypeptides. Thepolynucleotides can be administered in a gene delivery vehicle or byinserting into a host cell which in turn recombinantly transcribes,translates and processed the encoded polypeptide. Isolated host cellscontaining the polynucleotides of this invention in a pharmaceuticallyacceptable carrier can therefore combined with appropriate and effectiveamount of an adjuvant, cytokine or co-stimulatory molecule for aneffective vaccine regimen. In one embodiment, the host cell is an APCsuch as a dendritic cell. The host cell can be further modified byinserting of a polynucleotide coding for an effective amount of eitheror both a cytokine and/or a co-stimulatory molecule.

The methods of this invention can be further modified byco-administering an effective amount of a cytokine or co-stimulatorymolecule to the subject.

This invention also provides compositions containing any of theabove-mentioned proteins, polypeptides, polynucleotides, vectors, cells,antibodies and fragments thereof, and an acceptable solid or liquidcarrier. When the compositions are used pharmaceutically, they arecombined with a “pharmaceutically acceptable carrier” for diagnostic andtherapeutic use. These compositions also can be used for the preparationof medicaments for the diagnosis and treatment of diseases such ascancer.

The following materials and methods are intended to illustrate, but notlimit this invention and to illustrate how to make and use theinventions described above.

Materials and Methods

Production of the Polypeptides of the Invention

Most preferably, isolated peptides of the present invention can besynthesized using an appropriate solid state synthetic procedure.Steward and Young, SOLID PHASE PEPTIDE SYNTHESIS, Freemantle, SanFrancisco, Calif. (1968). A preferred method is the Merrifield process.See, Merrifield (1967) Recent Progress in Hormone Res. 23:451. Theantigenic activity of these peptides may conveniently be tested using,for example, the assays as described herein.

Once an isolated peptide of the invention is obtained, it may bepurified by standard methods including chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification. For immuno-affinity chromatography, an epitope may beisolated by binding it to an affinity column comprising antibodies thatwere raised against that peptide, or a related peptide of the invention,and were affixed to a stationary support.

Alternatively, affinity tags such as hexa-His (Invitrogen), Maltosebinding domain (New England Biolabs), influenza coat sequence (Kolodziejet al. (1991) Meth. Enzymol. 194:508–509), and glutathione-S-transferasecan be attached to the peptides of the invention to allow easypurification by passage over an appropriate affinity column. Isolatedpeptides can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance, and x-ray crystallography.

Also included within the scope of the invention are antigenic peptidesthat are differentially modified during or after translation, e.g., byphosphorylation, glycosylation, cross-linking, acylation, proteolyticcleavage, linkage to an antibody molecule, membrane molecule or otherligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285–320).

Isolation, Culturing and Expansion of APCs, Including Dendritic Cells

The following is a brief description of two fundamental approaches forthe isolation of APC. These approaches involve (1) isolating bone marrowprecursor cells (CD34⁺) from blood and stimulating them to differentiateinto APC; or (2) collecting the precommitted APCs from peripheral blood.In the first approach, the patient must be treated with cytokines suchas GM-CSF to boost the number of circulating CD34⁺ stem cells in theperipheral blood.

The second approach for isolating APCs is to collect the relativelylarge numbers of precommitted APCs already circulating in the blood.Previous techniques for isolating committed APCs from human peripheralblood have involved combinations of physical procedures such asmetrizamide gradients and adherence/non-adherence steps (Freudenthal P.S. et al. (1990) Proc. Natl. Acad. Sci. USA 87:7698–7702); Percollgradient separations (Mehta-Damani et al. (1994) J. Immunol.153:996–1003); and fluorescence activated cell sorting techniques(Thomas R. et al. (1993) J. Immunol. 151:6840–6852).

One technique for separating large numbers of cells from one another isknown as countercurrent centrifugal elutriation (CCE). In thistechnique, cells are subject to simultaneous centrifugation and awashout stream of buffer that is constantly increasing in flow rate. Theconstantly increasing countercurrent flow of buffer leads to fractionalcell separations that are largely based on cell size.

In one aspect of the invention, the APC are precommitted or maturedendritic cells which can be isolated from the white blood cell fractionof a mammal, such as a murine, simian or a human (See, e.g., WO96/23060). The white blood cell fraction can be from the peripheralblood of the mammal. This method includes the following steps: (a)providing a white blood cell fraction obtained from a mammalian sourceby methods known in the art such as leukophoresis; (b) separating thewhite blood cell fraction of step (a) into four or more subfractions bycountercurrent centrifugal elutriation; (c) stimulating conversion ofmonocytes in one or more fractions from step (b) to dendritic cells bycontacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSFand IL-4, (d) identifying the dendritic cell-enriched fraction from step(c); and (e) collecting the enriched fraction of step (d), preferably atabout 4° C. One way to identify the dendritic cell-enriched fraction isby fluorescence-activated cell sorting. The white blood cell fractioncan be treated with calcium ionophore in the presence of othercytokines, such as recombinant (rh) rhIL-12, rhGM-CSF, or rhIL-4. Thecells of the white blood cell fraction can be washed in buffer andsuspended in Ca⁺⁺/Mg⁺⁺ free media prior to the separating step. Thewhite blood cell fraction can be obtained by leukapheresis. Thedendritic cells can be identified by the presence of at least one of thefollowing markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absenceof the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20.Monoclonal antibodies specific to these cell surface markers arecommercially available.

More specifically, the method requires collecting an enriched collectionof white cells and platelets from leukapheresis that is then furtherfractionated by countercurrent centrifugal elutriation (CCE) (AbrahamsenT. G. et al. (1991) J. Clin. Apheresis. 6:48–53). Cell samples areplaced in a special elutriation rotor. The rotor is then spun at aconstant speed of, for example, 3000 rpm. Once the rotor has reached thedesired speed, pressurized air is used to control the flow rate ofcells. Cells in the elutriator are subjected to simultaneouscentrifugation and a washout stream of buffer that is constantlyincreasing in flow rate. This results in fractional cell separationsbased largely but not exclusively on differences in cell size.

Quality control of APC and more specifically DC collection andconfirmation of their successful activation in culture is dependent upona simultaneous multi-color FACS analysis technique which monitors bothmonocytes and the dendritic cell subpopulation as well as possiblecontaminant T lymphocytes. It is based upon the fact that DCs do notexpress the following markers: CD3 (T cell); CD14 (monocyte); CD16, 56,57 (NK/LAK cells); CD19, 20 (B cells). At the same time, DCs do expresslarge quantities of HLA-DR, significant HLA-DQ and B7.2 (but little orno B7.1) at the time they are circulating in the blood (in addition theyexpress Leu M7 and M9, myeloid markers which are also expressed bymonocytes and neutrophils).

When combined with a third color reagent for analysis of dead cells,propidium iodide (PI), it is possible to make positive identification ofall cell subpopulations (see Table 1):

TABLE 1 FACS analysis of fresh peripheral cell subpopulations Color #1Cocktail Color #2 Color #3 3/14/16/19/20/56/57 HLA-DR PI Live Dendriticcells Negative Positive Negative Live Monocytes Positive PositiveNegative Live Neutrophils Negative Negative Negative Dead Cells VariableVariable Positive Additional markers can be substituted for additionalanalysis: Color #1: CD3 alone, CD14 alone, etc.; Leu M7 or Leu M9;anti-Class I, etc. Color #2: HLA-DQ, B7.1, B7.2, CD25 (IL2r), ICAM,LFA-3, etc.

The goal of FACS analysis at the time of collection is to confirm thatthe DCs are enriched in the expected fractions, to monitor neutrophilcontamination, and to make sure that appropriate markers are expressed.This rapid bulk collection of enriched DCs from human peripheral blood,suitable for clinical applications, is absolutely dependent on theanalytic FACS technique described above for quality control. If need be,mature DCs can be immediately separated from monocytes at this point byfluorescent sorting for “cocktail negative” cells. It may not benecessary to routinely separate DCs from monocytes because, as will bedetailed below, the monocytes themselves are still capable ofdifferentiating into DCs or functional DC-like cells in culture.

Once collected, the DC rich/monocyte APC fractions (usually 150 through190) can be pooled and cryopreserved for future use, or immediatelyplaced in short term culture.

Alternatively, others have reported a method for upregulating(activating) dendritic cells and converting monocytes to an activateddendritic cell phenotype. This method involves the addition of calciumionophore to the culture media convert monocytes into activateddendritic cells. Adding the calcium ionophore A23187, for example, atthe beginning of a 24–48 hour culture period resulted in uniformactivation and dendritic cell phenotypic conversion of the pooled“monocyte plus DC” fractions: characteristically, the activatedpopulation becomes uniformly CD14 (Leu M3) negative, and upregulatesHLA-DR, HLA-DQ, ICAM-1, B7.1, and B7.2. Furthermore, this activated bulkpopulation functions as well on a small numbers basis as a furtherpurified.

Specific combination(s) of cytokines have been used successfully toamplify (or partially substitute) for the activation/conversion achievedwith calcium ionophore: these cytokines include but are not limited topurified or recombinant (“rh”) rhGM-CSF, rhIL-2, and rhIL-4. Eachcytokine when given alone is inadequate for optimal upregulation.

Presentation of Antigen to the APC

For purposes of immunization, the antigenic peptides (SEQ ID NOs: 3, 5,7, 9, 11 and 13) can be delivered to antigen-presenting cells asprotein/peptide or in the form of cDNA encoding the protein/peptide.Antigen-presenting cells (APCs) can consist of dendritic cells (DCs),monocytes/macrophages, B lymphocytes or other cell type(s) expressingthe necessary MHC/co-stimulatory molecules. The methods described belowfocus primarily on DCs which are the most potent, preferred APCs.

Pulsing is accomplished in vitro/ex vivo by exposing APCs to theantigenic protein or peptide(s) of this invention. The protein orpeptide(s) are added to APCs at a concentration of 1–10 μm forapproximately 3 hours. Pulsed APCs can subsequently be administered tothe host via an intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

Protein/peptide antigen can also be delivered in vivo with adjuvant viathe intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

Paglia et al. (1996) J. Exp. Med. 183:317–322 has shown that APCincubated with whole protein in vitro were recognized by MHC classI-restricted CTLs, and that immunization of animals with these APCs ledto the development of antigen-specific CTLs in vivo. In addition,several different techniques have been described which lead to theexpression of antigen in the cytosol of APCs, such as DCs. These include(1) the introduction into the APCs of RNA isolated from tumor cells, (2)infection of APCs with recombinant vectors to induce endogenousexpression of antigen, and (3) introduction of tumor antigen into the DCcytosol using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med.184:465–472; Rouse et al. (1994) J. Virol. 68:5685–5689; and Nair et al.(1992) J. Exp. Med. 175:609–612).

Foster Antigen Presenting Cells

Foster antigen presenting cells are particularly useful as target cells.Foster APCs are derived from the human cell line 174×CEM.T2, referred toas T2, which contains a mutation in its antigen processing pathway thatrestricts the association of endogenous peptides with cell surface MHCclass I molecules (Zweerink et al. (1993) J. Immunol. 150:1763–1771).This is due to a large homozygous deletion in the MHC class II regionencompassing the genes TAP1, TAP2, LMP1, and LMP2, which are requiredfor antigen presentation to MHC class I-restricted CD8⁺ CTLs. In effect,only “empty” MHC class I molecules are presented on the surface of thesecells. Exogenous peptide added to the culture medium binds to these MHCmolecules provided that the peptide contains the allele-specific bindingmotif. These T2 cells are referred to herein as “foster” APCs. They canbe used in conjunction with this invention to present antigen(s).

Transduction of T2 cells with specific recombinant MHC alleles allowsfor redirection of the MHC restriction profile. Libraries tailored tothe recombinant allele will be preferentially presented by them becausethe anchor residues will prevent efficient binding to the endogenousallele.

High level expression of MHC molecules makes the APC more visible to theCTLs. Expressing the MHC allele of interest in T2 cells using a powerfultranscriptional promoter (e.g., the CMV promoter) results in a morereactive APC (most likely due to a higher concentration of reactiveMHC-peptide complexes on the cell surface).

Immunogenicity Assays.

The immunogenicity of invention ligands can be determined by well knownmethodologies including, but not limited to those exemplified below. Inone embodiment, such methodology may be employed to compare an alteredligand of the invention with the corresponding native ligand. Forexample, an altered ligand may be considered “more active” if itcompares favorably with the activity of the native ligand in any one ofthe following assays. For some purposes, one skilled in the art willselect an immunogenic ligand which displays more activity than anotherimmunogenic ligand, i.e., for treatment and/or diagnostic purposes.However, for some applications, the use of an immunogenic ligand whichis comparable with the native ligand will be suitable. In othersituations, it may be desirable to utilize an immunogenic ligand whichis less active. It has been suggested that such levels of activitypositively correlate with the level of immunogenicity.

⁵¹Cr-release lysis assay. Lysis of peptide-pulsed ⁵¹Cr-labeled targetsby antigen-specific T cells can be compared for target cells pulsed witheither the native or altered ligands. Functionally enhanced ligands willshow greater lysis of targets as a function of time. The kinetics oflysis as well as overall target lysis at a fixed timepoint (e.g., 4hours) may be used to evaluate ligand performance. (Ware C. F. et al.(1983) J. Immunol. 131:1312).

Cytokine-release assay. Analysis of the types and quantities ofcytokines secreted by T cells upon contacting ligand-pulsed targets canbe a measure of functional activity. Cytokines can be measured by ELISAor ELISPOT assays to determine the rate and total amount of cytokineproduction. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160:181;Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).

In vitro T cell education. The ligands of the invention can be comparedto the corresponding native ligand for the ability to elicitligand-reactive T cell populations from normal donor or patient-derivedPBMC. In this system, elicited T cells can be tested for lytic activity,cytokine-release, polyclonality, and cross-reactivity to the nativeligand. (Parkhurst M. R. et al. (1996) J. Immunol. 157:2539).

Transgenic animal models. Immunogenicity can be assessed in vivo byvaccinating HLA transgenic mice with either the ligands of the inventionor the native ligand and determining the nature and magnitude of theinduced immune response. Alternatively, the hu-PBL-SCID mouse modelallows reconstitution of a human immune system in a mouse by adoptivetransfer of human PBL. These animals may be vaccinated with the ligandsand analyzed for immune response as previously mentioned. (Shirai M. etal. (1995) J. Immunol. 154:2733; Mosier D. E. et al. (1993) Proc. Natl.Acad. Sci. USA 90:2443).

Proliferation. T cells will proliferate in response to reactive ligands.Proliferation can be monitored quantitatively by measuring, for example,³-thymidine uptake. (Caruso A. et al. (1997) Cytometry 27:71).

Tetramer staining. MHC tetramers can be loaded with individual ligandsand tested for their relative abilities to bind to appropriate effectorT cell populations. (Altman J. D. et al. (1996) Science274(5284):94–96).

MHC Stabilization. Exposure of certain cell lines such as T2 cells toHLA-binding ligands results in the stabilization of MHC complexes on thecell surface. Quantitation of MHC complexes on the cell surface has beencorrelated with the affinity of the ligand for the HLA allele that isstabilized. Thus, this technique can determine the relative HLA affinityof ligand epitopes. (Stuber G. et al. (1995) Int. Immunol. 7:653).

MHC competition. The ability of a ligand to interfere with thefunctional activity of a reference ligand and its cognate T celleffectors is a measure of how well a ligand can compete for MHC binding.Measuring the relative levels of inhibition is an indicator of MHCaffinity. (Feltkamp M. C. et al. (1995) Immunol. Lett. 47:1).

Primate models. A recently described non-human primate (chimpanzee)model system can be utilized to monitor in vivo immunogenicities ofHLA-restricted ligands. It has been demonstrated that chimpanzees shareoverlapping MHC-ligand specificities with human MHC molecules thusallowing one to test HLA-restricted ligands for relative in vivoimmunogenicity. (Bertoni R. et al. (1998) J. Immunol. 161:4447).

Monitoring TCR Signal Transduction Events. Several intracellular signaltransduction events (e.g., phosphorylation) are associated withsuccessful TCR engagement by MHC-ligand complexes. The qualitative andquantitative analysis of these events have been correlated with therelative abilities of ligands to activate effector cells through TCRengagement. (Salazar E. et al. (2000) Int. J. Cancer 85:829; Isakov N.et al. (1995) J. Exp. Med. 181:375).

Expansion of Immune Effector Cells

The present invention makes use of these APCs to stimulate production ofan enriched population of antigen-specific immune effector cells. Theantigen-specific immune effector cells are expanded at the expense ofthe APCs, which die in the culture. The process by which naïve immuneeffector cells become educated by other cells is described essentiallyin Coulie (1997) Molec. Med. Today 3:261-268.

The APCs prepared as described above are mixed with naïve immuneeffector cells. Preferably, the cells may be cultured in the presence ofa cytokine, for example IL-2. Because dendritic cells secrete potentimmunostimulatory cytokines, such as IL-12, it may not be necessary toadd supplemental cytokines during the first and successive rounds ofexpansion. In any event, the culture conditions are such that theantigen-specific immune effector cells expand (i.e., proliferate) at amuch higher rate than the APCs. Multiple infusions of APCs and optionalcytokines can be performed to further expand the population ofantigen-specific cells.

In one embodiment, the immune effector cells are T cells. In a separateembodiment, the immune effector cells can be genetically modified bytransduction with a transgene coding for example, IL-2, IL-11 or IL-13.Methods for introducing transgenes in vitro, ex vivo and in vivo arewell known in the art. See Sambrook et al. (1989) supra.

Vectors Useful in Genetic Modifications

In general, genetic modifications of cells employed in the presentinvention are accomplished by introducing a vector containing apolypeptide or transgene encoding a heterologous or an altered antigen.A variety of different gene transfer vectors, including viral as well asnon-viral systems can be used. Viral vectors useful in the geneticmodifications of this invention include, but are not limited toadenovirus, adeno-associated virus vectors, retroviral vectors andadeno-retroviral chimeric vectors. APC and immune effector cells can bemodified using the methods described below or by any other appropriatemethod known in the art.

Construction of Recombinant Adenoviral Vectors or Adeno-Associated VirusVectors

Adenovirus and adeno-associated virus vectors useful in the geneticmodifications of this invention may be produced according to methodsalready taught in the art. See, e.g., Karlsson et al. (1986) EMBO J.5:2377; Carter (1992) Curr. Op. Biotechnol. 3:533–539; Muzcyzka (1992)Current Top. Microbiol. Immunol. 158:97–129; GENE TARGETING: A PRACTICALAPPROACH (1992) ed. A. L. Joyner, Oxford University Press, N.Y.).Several different approaches are feasible. Preferred is thehelper-independent replication deficient human adenovirus system.

The recombinant adenoviral vectors based on the human adenovirus 5(McGrory, W. J. et al. (1988) Virology 163:614–617) are missingessential early genes from the adenoviral genome (usually E1A/E1B), andare therefore unable to replicate unless grown in permissive cell linesthat provide the missing gene products in trans. In place of the missingadenoviral genomic sequences, a transgene of interest can be cloned andexpressed in cells infected with the replication deficient adenovirus.Although adenovirus-based gene transfer does not result in integrationof the transgene into the host genome (less than 0.1%adenovirus-mediated transfections result in transgene incorporation intohost DNA), and therefore is not stable, adenoviral vectors can bepropagated in high titer and transfect non-replicating cells. Human 293cells, which are human embryonic kidney cells transformed withadenovirus E1A/E1B genes, typify useful permissive cell lines. However,other cell lines which allow replication-deficient adenoviral vectors topropagate therein can be used, including HeLa cells.

Additional references describing adenovirus vectors and other viralvectors which could be used in the methods of the present inventioninclude the following: Horwitz M. S. ADENOVIRIDAE AND THEIR REPLICATION,in Fields B. et al. (eds.) VIROLOGY, Vol. 2, Raven Press New York, pp.1679–1721 (1990); Graham F. et al. pp. 109–128 in METHODS IN MOLECULARBIOLOGY, Vol. 7: GENE TRANSFER AND EXPRESSION PROTOCOLS, Murray E. (ed.)Humana Press, Clifton, N.J. (1991); Miller N. et al. (1995) FASEB J.9:190–199; Schreier H. (1994) Pharmaceutica Acta Helvetiae 68:145–159;Schneider and French (1993) Circulation 88:1937–1942; Curiel D. T. etal.(1992) Hum. Gene Ther. 3:147–154; Graham F. L. et al. WO 95/00655 (5Jan. 1995); Falck-Pedersen E. S. WO 95/16772 (22 Jun. 1995); Denefle P.et al. WO 95/23867 (8 Sep. 1995); Haddada H. et al. WO 94/26914 (24 Nov.1994); Perricaudet M. et al. WO 95/02697 (26 Jan. 1995); Zhang W. et al.WO 95/25071 (12 Oct. 1995). A variety of adenovirus plasmids are alsoavailable from commercial sources, including, e.g., Microbix Biosystemsof Toronto, Ontario (see, e.g., Microbix Product Information Sheet:Plasmids for Adenovirus Vector Construction, 1996). See also, the papersby Vile et al. (1997) Nature Biotechnology 15:840–841; and Feng et al.(1997) Nature Biotechnology 15:866–870, describing the construction anduse of adeno-retroviral chimeric vectors that can be employed forgenetic modifications.

Additional references describing AAV vectors that could be used in themethods of the present invention include the following: Carter B.HANDBOOK OF PARVOVIRUSES, Vol. I, pp. 169–228, 1990; Bems, VIROLOGY, pp.1743–1764 (Raven Press 1990); Carter B. (1992) Curr. Opin. Biotechnol.3:533–539; Muzyczka N. (1992) Current Topics in Micro. and Immunol,158:92–129; Flotte T. R. et al. (1992) Am. J. Respir. Cell Mol. Biol.7:349–356; Chattedjee et al. (1995) Ann. NY Acad. Sci. 770:79–90; FlotteT. R. et al. WO 95/13365 (18 May 1995); Trempe J. P. et al., WO 95/13392(18 May 1995); Kotin R.(1994) Hum. Gene Ther. 5:793–801; Flotte T. R. etal. (1995) Gene Therapy 2:357–362; Allen J. M. WO 96/17947 (13 Jun.1996); and Du et al. (1996) Gene Therapy 3:254–261.

APCs can be transduced with viral vectors encoding a relevantpolypeptide(s). The most common viral vectors include recombinantpoxviruses such as vaccinia and fowlpox virus (Bronte et al. (1997)Proc. Natl. Acad. Sci. USA 94:3183–3188; Kim et al. (1997) J.Immunother. 20:276–286) and, preferentially, adenovirus (Arthur et al.(1997) J. Immunol. 159:1393–1403; Wan et al. (1997) Human Gene Therapy8:1355–1363; Huang et al. (1995) J. Virol. 69:2257–2263). Retrovirusalso may be used for transduction of human APCs (Marin et al. (1996) J.Virol. 70:2957–2962).

In vitro/ex vivo, exposure of human DCs to adenovirus (Ad) vector at amultiplicity of infection (MOI) of 500 for 16–24 h in a minimal volumeof serum-free medium reliably gives rise to transgene expression in90–100% of DCs. The efficiency of transduction of DCs or other APCs canbe assessed by immunofluorescence using fluorescent antibodies specificfor the tumor antigen being expressed (Kim et al. (1997) J. Immunother.20:276–286). Alternatively, the antibodies can be conjugated to anenzyme (e.g., HRP) giving rise to a colored product upon reaction withthe substrate. The actual amount of antigenic polypeptides beingexpressed by the APCs can be evaluated by ELISA.

Transduced APCs can subsequently be administered to the host via anintravenous, subcutaneous, intranasal, intramuscular or intraperitonealroute of delivery.

In vivo transduction of DCs, or other APCs, can be accomplished byadministration of Ad (or other viral vectors) via different routesincluding intravenous, intramuscular, intranasal, intraperitoneal orcutaneous delivery. The preferred method is cutaneous delivery of Advector at multiple sites using a total dose of approximately1×10¹⁰–1×10¹² i.u. Levels of in vivo transduction can be roughlyassessed by co-staining with antibodies directed against APC marker(s)and the TAA being expressed. The staining procedure can be carried outon biopsy samples from the site of administration or on cells fromdraining lymph nodes or other organs where APCs (in particular DCs) mayhave migrated (Condon et al. (1996) Nature Med. 2:1122–1128 and Wan etal. (1997) Hum. Gene Ther. 8:1355–1363). The amount of antigen beingexpressed at the site of injection or in other organs where transducedAPCs may have migrated can be evaluated by ELISA on tissue homogenates.

Although viral gene delivery is more efficient, DCs can also betransduced in vitro/ex vivo by non-viral gene delivery methods such aselectroporation, calcium phosphate precipitation or cationiclipid/plasmid DNA complexes (Arthur et al. (1997) Cancer Gene Ther.4:17–25). Transduced APCs can subsequently be administered to the hostvia an intravenous, subcutaneous, intranasal, intramuscular orintraperitoneal route of delivery.

In vivo transduction of DCs, or other APCs, can potentially beaccomplished by administration of cationic lipid/plasmid DNA complexesdelivered via the intravenous, intramuscular, intranasal,intraperitoneal or cutaneous route of administration. Gene gun deliveryor injection of naked plasmid DNA into the skin also leads totransduction of DCs (Condon et al. (1996) Nature Med. 2:1122–1128; Razet al (1994) Proc. Natl. Acad. Sci. USA 91:9519–9523). Intramusculardelivery of plasmid DNA may also be used for immunization (Rosato et al.(1997) Hum. Gene Ther. 8:1451–1458.)

The transduction efficiency and levels of transgene expression can beassessed as described above for viral vectors.

Adoptive Immunotherapy and Vaccines

The expanded populations of antigen-specific immune effector cells ofthe present invention also find use in adoptive immunotherapy regimesand as vaccines.

Adoptive immunotherapy methods involve, in one aspect, administering toa subject a substantially pure population of educated, antigen-specificimmune effector cells made by culturing naïve immune effector cells withAPCs as described above. Preferably, the APCs are dendritic cells.

In one embodiment, the adoptive immunotherapy methods described hereinare autologous. In this case, the APCs are made using parental cellsisolated from a single subject. The expanded population also employs Tcells isolated from that subject. Finally, the expanded population ofantigen-specific cells is administered to the same patient.

In a further embodiment, APCs or immune effector cells are administeredwith an effective amount of a stimulatory cytokine, such as IL-2 or aco-stimulatory molecule.

The agents identified herein as effective for their intended purpose canbe administered to subjects having cell expressing CMV pp65 antigen aswell as or in addition to individuals susceptible to or at risk. Whenthe agent is administered to a subject such as a mouse, a rat or a humanpatient, the agent can be added to a pharmaceutically acceptable carrierand systemically or topically administered to the subject. To determinepatients that can be beneficially treated, a tumor regression can beassayed. Therapeutic amounts can be empirically determined and will varywith the pathology being treated, the subject being treated and theefficacy and toxicity of the therapy.

Administration in vivo can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with thecomposition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents can be found below.

The agents and compositions of the present invention can be used in themanufacture of medicaments and for the treatment of humans and otheranimals by administration in accordance with conventional procedures,such as an active ingredient in pharmaceutical compositions.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including nasal, topical (including transdermal, aerosol,buccal and sublingual), parental (including subcutaneous, intramuscular,intravenous and intradermal) and pulmonary. It will also be appreciatedthat the preferred route will vary with the condition and age of therecipient, and the disease being treated.

The preceding discussion and examples are intended merely to illustratethe art. As is apparent to one of skill in the art, variousmodifications can be made to the above without departing from the spiritand scope of this invention.

1. An isolated compound having the structure of SEQ ID NO.5.